One of the most widely employed methods of synthesizing oligonucleotides is known as the phosphate triester method. The phosphate triester method can be employed in solution, and generally involves coupling of a protected nucleoside 3'-phosphate with another protected nucleoside having a 5'-hydroxyl group. The coupled reaction product is typically isolated/purified chromatographically and one of the protecting groups is removed to yield a dimer block. The dimer block can then be coupled with other selected oligomeric blocks having an unprotected 3'-phosphate or 5'-hydroxyl terminus to yield oligomers having desired nucleoside sequences.
The phosphate triester method for oligonucleotide synthesis can also be readily adapted for solid phase reaction conditions. Thus a 5'-O-protected deoxyribonucleoside, for example, can be covalently attached to a solid support, such as polystyrene, cellulose, or silica gel, and subjected to a sequence of reaction conditions which first effects removal of the 5'-O-protecting group and thereafter couples the support bound nucleoside to other nucleosides. Repeating the process with selected reactive nucleosides, dimer blocks, or oligonucleotide intermediates enables extension of the nucleotide chain to a predetermined length/composition. In the final step the oligonucleotide is cleaved from the solid support and subjected to conventional purification techniques.
The present invention allows application of such solid phase synthesis procedures to the synthesis of phosphorodithioate oligonucleotide analogs.
Matsukura et al. , Proc. Natl. Acad. Sci., Vol. 84, pp. 7706-7710, 1987, have shown that phosphorothioate analogs of oligonucleotides are effective inhibitors of HIV replication and are cytopathic to virally infected T cells. They exhibit antiviral activity in vivo by binding with either the RNA template derived from a virus or duplex DNA derived from a virus that has integrated into the genome of the host. Both complementary "antisense" oligonucleotide phosphorothioates and homooligomer analogs exhibited potent anti-HIV activity. The most effective analog was reported to be a 28-mer oligodeoxycytidine phosphorothioate (S-dC.sub.28) which exhibited anti-HIV activity at 1 .mu.M concentration and inhibited de novo DNA synthesis. The authors reported that the S-analogs of deoxyribonucleotides (phosphorothioates) showed no significant degradation over a period of weeks in their cytopathic assay and during incubation in human serum at 37.degree. C. Hydrolysis of normal oligonucleotides indicated a half-life of .about.17 hr in the in vitro assay. The S-analogs also showed good permeability to the target cells. Using .sup.35 S-labeled phosphorothioates, S-dC.sub.28 showed significant amounts of radioactivity in the immortalized T4.sup.+ ATH8 and H9 cells within several minutes.
Notably, however, the phosphorothioate linkages of oligonucleotide analogs as disclosed by Matsukura et al. are chiral structures. In the best of circumstances one might be able to achieve &gt;99% yields in the nucleotide chain assembly steps during solid-phase synthesis. Yet, if a coupling reaction proceeds with no stereospecificity, only 50% of the dimer product will have the correct stereochemistry. Thus, for example, in the synthesis of an n-mer having chiral phosphorothioate internucleosidic linkages, the best theoretical yield of diastereomerically pure n-mer, would be 1/2.sup.n-1 (&lt;3% for a 6-mer and &lt;1.0% yield for an 8-mer). This highlights the importance of avoiding phosphorothioate or other chiral centers in oligonucleotide analogs. Use of achiral internucleosidic linkages in the construction of nucleotide analogs not only avoids yield loss due to unwanted diastereomeric by-products, but also eliminates need for complex separation of diastereomers.
It is therefore an object of the present invention to provide a method for synthesis of achiral dithiophosphate oligonucleotide analogs.
It is a further object of the invention to provide novel monohalohydrocarbyl thiophosphoramidites as intermediates for the synthesis of achiral analogs of oligonucleotides.
Yet another object of the invention is the use of tetrazole or other acidic pKa nitrogenous compounds to catalyze the production of thiophosphite coupled nucleosides by the reaction of nucleoside 3'-O-thiophosphoramidites with nucleoside 5'-hydroxyl groups.
In accordance with the foregoing objectives a method is provided for the synthesis of intermediates useful for producing achiral phosphorodithioate analogs of oligonucleotides. The unprotected 3'-hydroxyl group of a nucleoside (hereinafter meaning nucleoside or deoxynucleoside) or oligonucleoside (that term hereinafter inclusive of oligonucleotides, oligodeoxynucleotides, and analogs thereof having internucleosidic linkages other than phosphate) is coupled with a halohydrocarbylthiophosphoramidite to provide a nucleoside 3'-O-hydrocarbylthiophosphoramidite. That product is reacted with a nucleoside or an oligonucleoside having an unprotected 5'-hydroxyl group and a protected 3'-hydroxyl group in the presence of a weak nitrogenous acid having a pKa equal to or greater to the pKa of 1H-tetrazole to provide nucleosides coupled through a 3', 5' hydrocarbyl thiophosphite linkage. Oxidation of that coupled oligonucleside analog with sulfur converts the thiophosphite to a dithioate triester coupled oligonucleotide. Deprotection of the 3'-hydroxyl group, removal of the hydrocarbyl moiety, and repetition of the synthesis scheme allows construction of a nucleoside oligomers having achiral phosphorodithioate internucleosidic linkages.
In a preferred embodiment of the present invention, a monochloro-N,N-dialkylaminohydrocarbylthiophosphine is used to form the reactive nucleoside 3'-O-hydrocarbylthiophosphoramidite. Most preferred mono-chloro-N,N-dialkylaminohydrocarbylthiophosphine intermediates are those wherein the N,N-dialkylamino group is N,N-diisopropyl, N,N-dimethyl, or morpholino, and the hydrocarbylthio group is methylthio, benzylthio, chlorobenzylthio, or dichlorobenzylthio.
The intermediate nucleoside 3'-O-thiophosphoramidite can be coupled by reaction with a nucleoside or oligonucleoside having an unprotected 5'-hydroxyl group and a protected 3'-hydroxyl group in the presence of 1H-tetrazole. Oxidation of the coupled oligonucleoside analog with sulfur in the presence of a tertiary amine base, preferably pyridine or 2,6-lutidine, followed by removal of the hydrocarbyl moiety through reaction with sulfur nucleophiles such as thiophenol provides dimers or oligomers having a phosphorodithioate internucleosidic linkage.
Deprotection of the 5'-hydroxyl group of the resulting oligomer and repetition of the aforedescribed synthesis scheme allows for facile synthesis of dithiophosphate coupled oligonucleotide analogs having a predetermined oligonucleotide sequence. Thus by using appropriate reaction sequences of oligodeoxynucleotide analogs (again, including oligodeoxynucleotide analogs) in a predetermined combination of phosphorodithioate and, for example, natural phosphate or other linkages can be synthesized.
Oligonucleotide analogs having a phosphorodithioate linkages are of potential use for both therapeutic and diagnostic applications. Phosphorodithioate linked oligonucleotides are isosteric and isopolar with normal phosphodiester linkages and are expected to have other biochemical and biophysical properties similar to natural DNA. Such DNA analogs are expected to be relatively nuclease resistant and easily derivatized with reporter groups, two very significant chemical properties important for numerous biochemical and biological applications. Therapeutically, phosphorodithioate oligonucleotide analogs can also be used as antisense agents directed against foreign or aberrant genetic elements such as virally derived nucleic acids or mRNA of oncogenic or other undesired genetic elements. Dithiophosphate analogs of ribozymes can also be produced that could catalytically cleave mRNA of viral, bacterial, or oncogenic origin in addition to mRNA derived from any other undesired genetic elements.